Methods in molecular biology
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The results of clinical studies on the value of preemptive analgesia are far from being unanimous. There are a number of potential problems related to preemptive analgesia that could lead to controversy regarding its clinical significance. The following potential problems are analyzed: (1) terminology, (2) approach to reveal the effect of preemptive analgesia, (3) verification of the direct pharmacological effect of a treatment, (4) partial preemptive effect in control, (5) intensity of noxious stimuli, (6) difference in a drug concentration between study groups during postoperative period, and (7) outcome measures.
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The retinol carrier retinol-binding protein (RBP) forms in blood a complex with the thyroid hormone carrier transthyretin (TTR). The interactions of retinoid-RBP complexes, as well as of unliganded RBP, with TTR can be investigated by means of fluorescence anisotropy. RBP represents the prototypic lipocalin, in the internal cavity of which the retinol molecule is accommodated. ⋯ The fluorescence anisotropy technique is also suitable to study the interaction of TTR with apoRBP and RBP in complex with non-fluorescent retinoids. In the latter cases, the fluorescence signal is provided by a fluorescent probe covalently linked to TTR rather than by RBP-bound retinol. We report here on the preparation of recombinant human RBP and TTR, the covalent labeling of TTR with the fluorescent dansyl probe, and fluorescence anisotropy titrations for RBP and TTR.
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The heat/capsaicin sensitization and intradermal capsaicin injection models are safe and noninvasive paradigms to generate stable, long-lasting, and reproducible injury capable of producing an area of both primary and secondary hyperalgesia. Risk of skin injury is substantially reduced since lower levels of thermal and chemical irritation produce long-lasting cutaneous hyperalgesia. ⋯ Unlike the heat/capsaicin sensitization model, intradermal capsaicin results in a brief painful stimulus followed by a long lasting area of secondary hyperalgesia. The intradermal injection of capsaicin results in a transient, intense stinging sensation at the site of injection (e.g. heat allodynia) followed by a persistent area of secondary tactile allodynia.
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Catechol-O-methyltransferase (COMT) is an enzyme that plays a key role in the modulation of catechol-dependent functions such as cognition, cardiovascular function, and pain processing. Recently, our group demonstrated that three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous position, are associated with experimental pain sensitivity and onset of temporomandibular joint disorder. In order to determine the functional mechanisms whereby these haplotypes contribute to pain processing, a series of in vitro experiments were performed. ⋯ Site-directed mutagenesis that eliminated the stable structure restored the amount of translated protein. These data provide the first demonstration that combinations of commonly observed alleles in the coding region of the human COMT gene can significantly affect the secondary structure of corresponding mRNA transcripts, which in turn leads to dramatic alterations in the translation efficiency of enzyme crucial for a variety of essential functions. The protocols applied to the study of these molecular genetic mechanisms are detailed herein.
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Inflammation is a complex, multiscale biological response to threats - both internal and external - to the body, which is also required for proper healing of injured tissue. In turn, damaged or dysfunctional tissue stimulates further inflammation. ⋯ We have suggested the concept of translational systems biology, defined as a focused application of computational modeling and engineering principles to pathophysiology primarily in order to revise clinical practice. This chapter reviews the existing, translational applications of computational simulations and related approaches as applied to inflammation.